Agent controlling the production of osteoclast formation regulator and method of screening substance capable of controlling the production of osteoclast formation regulator

ABSTRACT

A process for producing a polyether polymer by polymerizing a monomer having an oxirane group in the presence of a Lewis base having no active hydrogen such as, for example, a nitrile compound, a cyclic ether compound or an ester compound; and a polyether polymer produced by this process. Thus, a polyether polymer having a minimized amount of undesirable crosslinked product can be produced without reduction of polymerization activity.

TECHNICAL FIELD

The present invention relates to a novel agent for controlling theproduction of osteoclast-formation (or osteoclastogenesis) inhibitoryfactor, more particularly, an agent for controlling the production ofosteoclastogenesis inhibitory factor, which comprises a saccharide as aneffective ingredient, and a method for screening substances capable ofcontrolling the production of osteoclastogenesis inhibitory factor.

BACKGROUND ART

Osteoclastogenesis inhibitory factor also known as osteoprotegerin(abbreviated as “OPG” hereinafter) is known to be produced fromosteoblasts and organs such as liver, kidney, lung, and heart of mammalsand humans, and it has been highlighted in these days as an importantbone resorption inhibitor. Simonet W S et al. reported the gene forcoding OPG in Cell, Vol. 89, pp. 309-319 (1997), and this acceleratedthe study of OPG rapidly. It is said that OPG exists in blood, acts onthe bone metabolism systemically, and inhibits the formation ofosteoclast. Thus, as a means for normalizing mammals and humans fromabnormal osteoclast formation conditions, if the production level of OPGin mammals in such abnormal conditions could be controlled, theformation of osteoclast in abnormal conditions would be improved andmaintained in normal conditions, and the bone would be normalized andkept in healthy conditions.

However, there has been found almost no report on controlling theproduction of OPG. Under these circumstances, required is theestablishment of agents which may control the production of OPG andwhich can be used safely, easily, and daily at users' homes withoutrestricting to their use in hospitals and clinics.

In view of these backgrounds, the first object of the present inventionis to establish an agent for controlling the production of OPG whichcontrols the production of OPG and which can be used safely, easily, anddaily at users' homes without restricting to their use in hospitals andclinics. The second object of the present invention is to provide amethod for screening substances, which can be incorporated into suchagent as an effective ingredient, promptly, easily, and accurately.

DISCLOSURE OF INVENTION

During energetically studying the physiological functions ofsaccharides, particularly, α,α-trehalose, α,β-trehalose, andβ,β-trehalose as non-reducing saccharides (these trehalose isomers aredesignated as “trehalose” throughout the specification, unless specifiedotherwise), the present inventors discovered that the culture ofepithelial cells of human small intestinal mucosae in the presence oftrehalose improved the production level of OPG, and that, amongdifferent saccharides, there exist some saccharides which inhibit theproduction of OPG. Also, the present inventors discovered that culturingof an established cell line, which produces OPG in the presence oftrehalose, increased the production level of OPG in the culture. Basedon these findings, the present invention was made.

The present invention solves the above first object by providing anagent for controlling the production of OPG, which comprises a substancecapable of controlling the production of OPG.

The present invention solves the above second object by providing amethod for screening a substance capable of controlling the productionof OPG.

BRIEF EXPLANATION OF ACCOMPANYING DRAWING

FIG. 1 is a figure for the relative expression intensity of OPGcontained in each supernatant after 2-day cell culture in the presenceor the absence of different saccharides.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to an agent for controlling the productionof OPG, which comprises a saccharide as an effective ingredient, and toa method for screening substances capable of controlling the productionof OPG. The saccharides usable in the present invention include one ormore of the following saccharides alone or in an appropriatecombination: Monosaccharides such as D-glucose, D-mannose, D-galactose,D-arose, D-altrose, D-idose, D-arabinose, D-ribose, D-xylose, D-lyxose,D-fucose, D-fructose, D-talose, L-sorbose, D-tagatose, and D-psicose;disaccharides such as maltose, trehalose, sucrose, lactose, turanose,and rutinose; oligosaccharides of trisaccharides or higher such asmaltotriose and lactosucrose; and cyclic saccharides such astetrasaccharide having the structure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1},and cyclodextrins. In the present invention, among these saccharides,trehalose is particularly suitably used as a substance, which controlsthe production of OPG in mammals and humans, i.e., as a substance whichenhances the production of OPG. In the case of using trehalose as aneffective ingredient for the agent of the present invention, one or moreof isomers of trehalose, i.e., α,α-trehalose, α,β-trehalose, andβ,β-trehalose can be arbitrarily used in an effective amount. Theseisomers should not be restricted to homogeneous ones, and any trehalosecan be used independently of their preparation methods, purities,properties, and origins as long as other ingredients, coexisted with theisomers, do not hinder the action of trehalose.

As regards the process for producing trehalose usable in the presentinvention, detailed description thereof is omitted because the presentinvention does not relate to such a process in itself. However, when thefeasibility, production cost, and property of trehalose to be producedare weighed, preferably employed are the processes comprising a step ofsubjecting partial starch hydrolyzates to the actions of non-reducingsaccharide-forming enzyme and trehalose-releasing enzyme, disclosed inJapanese Patent Kokai Nos. 143,876/95, 213,283/95, 322,883/95,298,880/95, 66,187/96, 66,188/96, 336,388/96, and 84,586/96, applied forby the same applicant as the present invention. With these processes, ahigh quality trehalose can be easily produced in a higher yield fromstarches as a relatively cheap material. Examples of commercializedtrehalose products prepared by such processes are “TREHA®”, acrystalline trehalose powder having a trehalose content of 98% or more,commercialized by Hayashibara Shoji, Inc., Okayama, Japan; and“TREHASTAR®”, a trehalose syrup having a trehalose content of 28% ormore, commercialized by Hayashibara Shoji, Inc., Okayama, Japan.α,α-Trehalose can be also produced by subjecting maltose either to theaction of a maltose/trehalose converting enzyme disclosed in JapanesePatent Kokai Nos. 170,977/95, 263/96, or 149,980/96, applied for by thesame applicant as the present invention; or to the coaction ofconventional maltose phosphorylase and trehalose phosphorylase.

To produce α,β-trehalose, cyclomatodextrin glucanotransferase (CGTase),and β-galactosidase can be allowed to act in this order on a mixture oflactose and partial starch hydrolyzate, according to the methodsdisclosed in Japanese Patent Kokai Nos. 144,694/92 and 179,490/92,applied for by the same applicant as the present invention. While,β,β-trehalose can be produced by conventional chemical syntheses. Sincethe agent for controlling the production of OPG according to the presentinvention is orally or intubationally administered to intestinal tractsof subjects, the saccharides used for such purposes should not berestricted to those with the highest possible purity and also includethose with a relatively low purity, i.e., those which comprise thedesired saccharides and other ingredients that are unavoidably formed asby-products, depending or their preparation methods; those in a mixtureform of the desired saccharides and other appropriate ingredients whichdo not substantially hinder the action of the saccharides forcontrolling the production of OPG in mammals and humans.

The substances, which control the production of OPG, usable in thepresent invention include as a whole those which control the productionof OPG and can be selected by the later described method for screeningsuch substances. In the present invention, saccharides are exemplifiedas such substances screened by the above-mentioned method, however, anysubstance can be also used similarly as the above saccharides as long asthey are capable of controlling the production of OPG and orally orintubationally administered to mammals and humans. Accordingly, one ormore substances, that are selected by the screening method of thepresent invention, can be used as the effective ingredients for theagent for controlling the production of OPG of the present invention.

The agent for controlling the production of OPG according to the presentinvention can be in a single form of a substance capable of controllingthe production of OPG, or in a composition form comprising such asubstance and other ingredient(s). Usually, the agent of the presentinvention is provided in the form of a composition such as a food,health food, health supplement, or pharmaceutical, which can be orallyadministered or intubationally administered to subjects in the form of atube fed liquid diet or intubation clysis; a composition in the form ofa solution, suspension, emulsion, cream, paste, powder, granule, orother shaped-product with the desired form. When the composition is inthe form of a food product, materials and/or ingredients, which aregenerally used in food products, such as water, alcohols, amylaceoussubstances, proteins, fibers, saccharides, lipids, vitamins, minerals,flavors, colors, sweeteners, seasonings, stabilizers, antioxidants,antiseptics, viscosity-imparting agents, and fillers can be incorporatedinto the composition to ease the intake of the substances capable ofcontrolling the production of OPG. To the above-identified compositions,one or more of other materials for health supplements such as bifidbacteria, saccharides for promoting the growth of bifid bacteria, milkpowders, hydrolyzates of milk protein including casein calcium peptidesand casein phosphopeptides, lactoferrin, soybean isoflavone, blood meal,bone meal, shellfish meal, and coral meal. These food products may be inan orally administrable form or an intubationally administrable formsuch as those of tube fed liquid diets and intubation clyses.

In case the composition is in the form of a pharmaceutical, one or moreof carriers, excipients/adjuvants, diluents, and stabilizers are mixedwith other ingredients, and optionally further mixed with one or more ofcalcium agents such as calcium lactate, calcium glycerophosphate,calcium hydrogenphosphate, and calcium L-asparaginate, and othermedicaments such as analgesics, anti-inflamatories, active vitamin D,vitamin K preparations, calcitonin preparations, estrogen preparations,and anabolic hormone preparations. Varying depending on actual use, theagent of the present invention usually contains at least 0.1% (w/w) (“%(w/w)” is abbreviated as “%” hereinafter), preferably, at least 1% of asubstance(s) capable of controlling the production of OPG.

Now explaining the use of the agent of the present invention for humans,the agent is used to act on epithelial cells of human small intestinalmucosae and to effectively control the production of OPG in theepithelia cells, independently of its oral or incubationaladministration. Also, the agent of the present invention acts onepithelial cells of mammals other than human small intestinal mucosae,controls the production of OPG in the epithelia cells, and effectivelyinhibits the formation of osteoclast. Based on these, the agent of thepresent invention can be advantageously used as an agent for controllingthe production of OPG and also used to prevent, treat, or improvebone-related diseases such as bone fractures and cracks. Varyingdepending on the intended use, in the case of improving/promoting thehealth conditions of bone, the agent of the present invention is orallyadministered to subjects usually in the form of a food product. For thepurpose of relieving low-back-pain and arthralgia and improving thetherapeutic effect of the agent against bone damages/diseases, the agentis orally administered to subjects in the form of, for example, a foodproduct or a pharmaceutical such as a liquid, syrup, powder, granule,tablet, or capsule; or intubationally administered to subjects in theform of a liquid. The dose of the agent of the present invention isadministered to subjects at a dose, usually, of about 1 to 100g/adult/day, preferably, about 2 to 50 g/adult/day, daily or at afrequency of one to five shots per week. Varying depending on thepurpose of the agent to be used and the symptoms of humans or mammals tobe administered, the administration term is usually at least one month,preferably, at least three months, more preferably, at least six months.When saccharides are used as the effective ingredient of the agent ofthe present invention, they are most preferably applicable to practicingthe present invention because such saccharides are effectively used asenergy sources for living bodies and administrable for a relatively longperiod of time.

The method for screening substances, which control the production of OPGaccording to the present invention, is explained below: The methodcomprises the steps of culturing an established cell line capable ofcontrolling the production of OPG in a nutrient culture medium, adding atest sample to the cell culture, culturing the cell line in the presenceof the test sample, assaying the production level of OPG released in theculture supernatant, comparing the production level of OPG with that ofcontrol, and judging the test sample to be a positive substance capableof controlling the production of OPG when the production level of OPGfor the test sample is higher or lower than that for a control system.The screening method has an advantage that it screens targetedsubstances which control the production of OPG, promptly, easily, andaccurately.

Preferable examples of such established cell lines are MG-63 cells, ATCCCRL-1427, an established cell line derived from human osteosarcoma; andFHs 74 Int cells, ATCC CCL-241, an established cell line derived from ahuman fetus small intestine. In practicing the present invention, theestablished cell lines usable in the present invention should not berestricted to the above-identified cell lines, and any cell lines can beused as long as they produce OPG. As the nutrient culture media used inculturing such cell lines, those free of saccharide are preferably used;MEM and RPMI 1640 media, which are free of saccharide and serum and inwhich the above cells can grow, are preferably used. In culturing theabove cells, they are suspended in a culture medium to give a final cellconcentration of 1×10³ to 1×10⁷ cells/ml, preferably, 5×10³ to 1×10⁶cells/ml, distributed to culture vessels having at least one or morewells, for example, those in the form of a plate having at least one ormore wells, cultured at 30 to 40 C, preferably, 35 to 38 C for 24 to 72hours in a 5% (v/v) CO₂ incubator, and washed several times with a freshpreparation of the same medium as used in the culture, followed byremoving each supernatant from the wells. To the remaining cells in eachwell was added a prescribed volume of a medium prepared by stepwiselydiluting a test sample with a fresh preparation of the same medium asused in the culture, and cultured for one to five days under the sameconditions as in the above culture. As a control, a system free of testsample was used. At a prescribed timing during cell culture, a portionof each culture medium and that for control were respectively sampled,followed by assaying the production level of OPG for each sample,comparing the production level of OPG for the test sample with that ofcontrol, and judging the test sample as a positive substance which cancontrol the production of OPG when the level of OPG for the test sampleis higher or lower than that for the control system.

The method for screening substances, which can control the production ofOPG, according to the present invention has a feature of screeningsubstances with such potential easily, effectively, and promptly. Alsothe method has advantageous features that it does not need anyexperimental animal and has beneficial convenience and satisfactorycost-performance in such a manner of being easily practiced even in arelatively small experimental/clinical laboratories.

The efficacy and the safety of the agent of the present invention isexplained with reference to the following experiments:

Experiment 1: Production Control of OPG by Substance Capable ofControlling the Production of OPG

Experiment 1-1: Preparation of Cell Culture

FHs 74 Int cells, ATCC CCL 241, were inoculated to 96-well plates, withD-MEM medium prepared by adding fetal calf serum, commercialized byGIBCO Bethesda Research Laboratories, Bethesda, Md., USA, to theserum-free culture medium shown in Table 1 below to give a finalconcentration of 10% (v/v), a final cell density of 1.2×10⁴ cells/well,and a final volume of 200 μl; and incubated at 37° C. for 48 hours in a5% CO₂ (V/V) incubator. After completion of the culture, the resultantcells were washed thrice (200 μl×3) with the serum-free D-MEM medium inTable 1. For a control group, using a fresh preparation of the samemedium as used in the above, the saccharide solutions of Nos. 1 to 7with the compositions shown in Table 2 were prepared, where thesaccharide solution of No. 1 consisted of 1 mg/ml of D-glucose with apurity of at least 99%, d.s.b., commercialized by Katayama ChemicalIndustries, Co., Ltd., Tokyo, Japan; the saccharide solution of No. 2, 1mg/ml of sucrose with a purity of at least 99%, d.s.b., commercializedby Wako Pure Chemical Industries, Ltd. Tokyo, Japan; the saccharidesolution of No. 3, 1 mg/ml of “MALTOSE HHH™”, with a purity of at least99%, d.s.b., commercialized by Hayashibara Biochemical Laboratories,Inc., Okayama, Japan; the saccharide solutions of No. 4, No. 5, and No.6, which were respectively 1 mg/ml, 0.2 mg/ml, and 0.04 mg/ml ofα,α-trehalose with a purity of at least 99%, d.s.b., commercialized byWako Pure Chemical Industries, Ltd., Tokyo, Japan; and the saccharidesolution of No. 7, 1 mg/ml of D-glucose with a purity of at least 99%,d.s.b., commercialized by Wako Pure Chemical Industries, Ltd., Tokyo,Japan. Two hundred microliters of each of the saccharide solutions ofNos.1 to 7 were placed in each well of the 96-well plates with thecells, and the cells were incubated at 37 C for two days in a 5% CO₂(v/v) incubator. The culture supernatant in each well was sampled andassayed for the production level of OPG by the Western Blotting methodas indicated below using a recombinant human OPG commercialized by R & DSystems Inc., MN, USA. In parallel, a cell culture system with nosaccharide, as a control group, was provided and cultured, and thesupernatant of the cell culture was sampled on day 2 after theinitiation of the cell culture and assayed for the production level ofOPG similarly as in the test group. TABLE 1 Composition of serum-freemedium: D-MEM Medium free of D-glucose, commercialized by GIBCO BethesdaResearch Laboratories, Bethesda, Maryland, USA MEM Non-essential aminoacid solution, commercialized by GIBCO Bethesda Research Laboratories,Bethesda, Maryland, USA MEM Sodium pyruvate solution, commercialized byGIBCO Bethesda Research Laboratories, Bethesda, Maryland, USA Sodiumselenite, in an amount of giving a final concentration of 20 nM,commercialized by Sigma Chemical Co., MO, USA Transferrin, in an amountof giving a final concentration of 5 μg/ml, commercialized by SigmaChemical Co., MO, USA Insulin, in an amount of giving a finalconcentration of 5 μg/ml, commercialized by Sigma Chemical Co., MO, USA

TABLE 2 Saccharide solution No. Saccharide Concentration 1 D-Glucose   1mg/ml 2 Sucrose   1 mg/ml 3 Maltose   1 mg/ml 4 α,α-Trehalose   1 mg/ml5 α,α-Trehalose  0.2 mg/ml 6 α,α-Trehalose 0.04 mg/ml 7 D-Glucose   1mg/ml + α,α-Trehalose   1 mg/mlExperiment 1-2: Western Blotting Method

Fifteen microliters of any of the cell culture supernatants of the testsolutions and the control solution, and five microliters ofdithiothreitol (DTT) solution for cell treatment were mixed and heatedat 99° C. for five minutes. Each of the resulting solutions with heattreatment was developed on SDS-polyacrylamide gel electrophoresis(SDS-PAGE) using multi-gel 4/20 commercialized by Daiichi Pure ChemicalsCo., Ltd., Tokyo, Japan; and the gels were transferred to PVDF membranescommercialized by Nihon Millipore Corporation, Tokyo, Japan. Theresulting PVDF membranes were blocked with a solution of “BLOCK ACE™”,commercialized by Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan, andcontacted with an anti-human OPG antibody as a primary antibody,commercialized by R & D Systems, MN, USA, and an HRP anti-goat IgGantibody, as a secondary antibody, commercialized by Sigma Chemical Co.,MO, USA; followed by detecting proteins reacted with these antibodies inaccordance with the method described in Experimental Medicine, specialedition, titled Handbook for novel recombinant technology, pp. 222-226(1999). The coloration was effected by using ECL reagent, commercializedby Amersham Pharmacia Biotechnology, IL, USA; exposing “HYPERFILM ECL™”;and measuring the expression intensity by using “IMAGE MASTER™”,commercialized by Amersham Biosciences, Tokyo, Japan. The results are inFIG. 1, where the production inhibitory level of OPG by saccharides isexpressed with a relative value (%) by regarding the expressionintensity of OPG for the control cell culture system as 100. As aresult, when α,α-trehalose was used alone or in combination withD-glucose, the expression intensity of OPG for the cells treated withsuch saccharides was higher than that for the control cell culturesystem, while the cells treated with D-glucose, maltose, and sucrose,the expression level of OPG for the cells was lower than that for thecontrol cell culture system. From these results, α,α-trehalose has anaction of promoting the production of OPG, while D-glucose, sucrose, andmaltose have an action of inhibiting the production of OPG. Whenα,α-trehalose and D-glucose were used in combination, D-glucose improvedthe action of α,α-trehalose to enhance the production of OPG.

Experiment 2: Method for Screening Substances Capable of Controlling theProduction of OPG

Similarly as in Experiment 1 except for using MG-63 cells, ATCC CRL1427, in place of FHs 74 Int cells, ATCC CCL 241, used in Experiment 1,the saccharide solutions of Nos. 1 to 7 with the compositions in Table 2were tested whether these saccharides control the production of OPG inMG-63 cells.

As a result, α,α-trehalose distinctly enhanced the production of OPG inthe cells compared with a control culture system free of saccharide,revealing that α,α-trehalose is a substance capable of distinctlyenhancing the production of OPG. Comparing with the control system freeof saccharide, D-glucose, maltose, and sucrose remarkably inhibited theproduction of OPG, revealing that these saccharides are substancescapable of distinctly inhibiting the production of OPG.

Although this experiment exemplified a method for screening differentsaccharides whether they have an ability of controlling the productionof OPG, such a method would be applicable to other natural and syntheticsubstances similarly as the above-identified saccharides, such asproteins, organic/inorganic compounds, extracts from animal and plantorigins, polysaccharides, hormones, and physiologically activesubstances to examine whether they have an ability of controlling theproduction of OPG.

Experiment 3: Acute Toxicity Test

In a physiological saline containing 5% gum arabic were dissolvedα,α-trehalose with a purity of at least 98%, d.s.b., commercialized byHayashibara Shoji, Inc., Okayama, Japan, and an adequate amount of anyof a special reagent grade of D-glucose, maltose, or sucrose with apurity of at least 99%, d.s.b., and the resulting solutions weresterilized in a usual manner. Either of the sterilized saccharidesolutions was administered with a syringe or intubationally administeredwith a stomach sonde to five groups of ddy mice, having 20 to 25 g bodyweight, consisting of five heads in each group. The mice were observedtheir conditions for seven days. As a result, no mouse died even whenadministered with 15 g/kg body weight of α,α-trehalose, D-glucose,maltose, or sucrose, independently of their administration routes. Theresult indicates that the agent of the present invention is safe evenwhen administered to humans and other mammals.

Experiment 4: Administration Test of Agent for Controlling theProduction of OPG

Eighteen healthy volunteers, aged 35 to 50-years-old, were divided intothree groups A, B, and C, consisting of three males and three females ineach group. Test solution 1 was prepared by dissolving “TREHA®”, acrystalline α,α-trehalose with a trehalose content of at least 98%,d.s.b., commercialized by Hayashibara Shoji, Inc., Okayama, Japan, in1,000 ml deionized water to give a concentration of 10% (w/v), andsterilized with a membrane filter. Test solution 2 was prepared bydissolving D-glucose with a purity of at least 99%, d.s.b.,commercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan, in1,000 ml of deionized water to give a concentration of 10% (w/v) andsterilized with a membrane filter. As a control solution, 1,000 ml ofdeionized water were sterilized with a membrane filter. These test andcontrol solutions were stored at about 10° C. in a refrigerator untilinitiating the test. The volunteers in the groups A to C were allowed tofast, except for water, from 10 p.m. at night just before initiating thetest, and 100 ml aliquots of the test solution 1, the test solution 2,and the control solution were administered to the groups A, B, and C,respectively, at 6 a.m. on the next day. Thereafter, all the volunteerswere allowed to stay indoors calmly and sampled their blood at six hoursafter the administrations. The sampled bloods were centrifuged at 35,000rpm for 10 min and quantified for OPG with “RS000805”, a product codenumber for ELISA system for assaying OPG commercialized by FunakoshiCo., Ltd., Tokyo. Japan. As a result, the production levels of OPG ofthe volunteers in the test group A, who were orally administered withα,α-trehalose as the test solution 1, were significantly higher thanthose with the test solution 2 and the control solution. The group Bwith D-glucose showed a significantly lower production level of OPG thanthat of the group C with the control solution.

After completion of the administration test, no volunteer, in the groupA with α,α-trehalose and the group B with D-glucose, complained aboutabnormality. The data of this experiment revealed that, when orallyadministered to living bodies, α,α-trehalose enhances the production ofOPG but D-glucose inhibits the production thereof.

The preferred embodiments according to the present invention areexplained with reference to the following examples:

EXAMPLE 1 Health Food

According to conventional manner, potatoes, which had been preserved at20° C. and a relative humidity of 85% for two weeks to self-assimilatereducing sugars, were washed with water, pealed, selected, and cut witha slicer into slices, 1.5 mm in thickness. After the slices were washedwith water to remove starch on their surfaces, drained water, and friedwith a salad oil at 170° C. for about five minutes, followed by removingthe salad oil. Using a salter, the fried products were uniformly sprayedwith a powdery seasoning comprising seven parts by weight of salt, threeparts by weight of “TREHA®”, a crystalline α,α-trehalose with atrehalose content of at least 98%, d.s.b., commercialized by HayashibaraShoji, Inc., Okayama, Japan, and an adequate amount of a spice. Theresulting products were transferred to a device for weighing andpackaging, weighed and packaged with the device, and sealed to obtain asnack-type food.

The product has a satisfactory flavor and taste, and it can beadvantageously used as a health food which acts on epithelial cells ofmammalian small intestinal mucosae, controls the production of OPG,effectively inhibits the formation of osteoclast relating to osteopathy,and maintains/promotes the health of bone, when orally administered tomammals.

EXAMPLE 2 Health Food

To 25 parts by weight of well kneaded butter were added 18 parts byweight of “TREHA®”, a crystalline α,α-trehalose with a trehalose contentof at least 98%, d.s.b., commercialized by Hayashibara Shoji, Inc.,Okayama, and 10 parts by weight of chicken eggs in this order, followedby mixing to obtain a creamy product. Forty-seven parts by weight ofsoft flour were added to the creamy product, and the resulting mixturewas kneaded and wrapped with a cloth and allowed to stand for 20 min.The resulting dough was shaped into a rod form, 3 cm in diameter,wrapped with a paraffin paper, and allowed to stand at 4° C. for twohours. The resulting rod-shaped dough was sliced in rounds, 5 mm inthickness, and the slices were placed on a plate with grease, baked inan oven at 170° C. for 10 min, applied with “TREHASTAR®”, a trehalosesyrup with an α,α-trehalose content of at least 28%, d.s.b.,commercialized by Hayashibara Shoji, Inc., Okayama, Japan, and furtherbaked at the same temperature as in the above for 10 min to obtain anicebox-type cookie.

The product has a satisfactory flavor and taste, and it can beadvantageously used as a health food that acts on epithelial cells ofmammalian small intestinal mucosae, controls the production of OPG,effectively inhibits the formation of osteoclast relating to osteopathy,and maintains/promotes the health of bone, when orally administered tomammals.

EXAMPLE 3 Health Food

Seven parts by weight of a freeze-dried tea extract, three parts byweight of α,β-trehalose, and three parts by weight of D-glucose weredissolved in water. The resulting solution was sprayed over 90 parts byweight of tea leaves which had been fermented and dried in a usualmanner. According to conventional manner, the tea leaves thus obtainedwere sieved, cut, dried for finish, subjected to a separator forremoving impurities, and packed with a Japanese paper in an amount oftwo grams.

Before drinking, the product is soaked for extraction in 180 ml of coldwater for about 10 min or of hot water heated to 90 to 100° C. for abouttwo min.

The product has a satisfactory flavor and taste, and it can beadvantageously used as a health food that acts on epithelial cells ofmammalian small intestinal mucosae, controls the production of OPG,effectively inhibits the formation of osteoclast relating to osteopathy,and maintains/promotes the health of bone, when orally administered tomammals.

EXAMPLE 4 Health Food

A coffee-type beverage was prepared by mixing in a usual manner 2.7parts by weight of “TETRUP®”, a maltotetraose syrup containing, on a drysolid basis, about 2% glucose, about 7% maltose, about 11% maltotriose,about 50% maltotetraose, and about 28% dextrin, commercialized byHayashibara Shoji, Inc., Okayama, Japan; seven parts by weight of“TREHASTAR®”, a trehalose syrup with an α,α-trehalose content of atleast 28%, d.s.b., commercialized by Hayashibara Shoji, Inc., Okayama,Japan; five parts by weight of a coffee extract, 2.2 parts by weight ofa whole milk powder, one part by weight of a skim milk powder, 0.04 partby weight of sucrose fatty acid ester, 0.06 part by weight of sodiumbicarbonate, and 82 parts by weight of water.

The product has a satisfactory flavor and taste, and it can beadvantageously used as a health food that acts on epithelial cells ofmammalian small intestinal mucosae, controls the production of OPG,effectively inhibits the formation of osteoclast relating to osteopathy,and maintains/promotes the health of bone, when orally administered tomammals.

EXAMPLE 5 Health Food

Fifty-five parts by weight of “TREHA®”, a crystalline α,α-trehalosepowder with an α,α-trehalose content of at least 98%, d.s.b.,commercialized by Hayashibara Shoji, Inc., Okayama, 40.5 parts by weightof corn starch, and 2.5 parts by weight of crystalline cellulose weremixed and then in a usual manner kneaded while spraying and droppingthereto a small amount of water, subjected to fluidized-bed granulation,pulverized, and sized to obtain a powder for tabletting. To the powderwas added two parts by weight of sucrose fatty acid ester, and themixture was mixed to homogeneity and tabletted by a tabletting machinewith a punch of 11 mm to obtain a tablet containing α,α-trehalose, about300 mg each.

The product has a satisfactory swallowability and degradability inintestines, and it can be advantageously used as a health food that actson epithelial cells of mammalian small intestinal mucosae, controls theproduction of OPG, effectively inhibits the formation of osteoclastrelating to osteopathy, and maintains/promotes the health of bone, whenorally administered to mammals.

EXAMPLE 6 Health Supplement

Thirty-nine parts by weight of “TREHA®”, a crystalline α,α-trehalosepowder with an α,α-trehalose content of at least 98%, d.s.b.,commercialized by Hayashibara Shoji, Inc., Okayama, 25 parts by weightof a natural coral powder, 12 parts by weight of a yogurt powder, 10parts by weight of guar gum, 1.9 parts by weight of2-O-α-D-glucopyranosyl-L-ascorbic acid, and 0.1 part by weight ofα-glycosyl hesperidin were in a usual manner kneaded while spraying anddropping to the contents an adequate amount of water, subjected tofluidized-bed granulation, pulverized, and sized to obtain a powder fortabletting. After homogeneously mixing the powder with three parts byweight of sucrose fatty acid ester as a gloss-imparting agent, theresulting mixture was tabletted by a tabletting machine with a punch of6 mm in diameter to obtain tablets, about 200 mg each.

The product is enriched with calcium and easily taken orally, and it canbe advantageously used as a health food that acts on epithelial cells ofmammalian small intestinal mucosae, controls the production of OPG,effectively inhibits the formation of osteoclast relating to osteopathy,and maintains/promotes the health of bone, when orally administered tomammals.

EXAMPLE 7 Health Supplement

Three parts by weight of “TREHA®”, a crystalline α,α-trehalose powderwith an α,α-trehalose content of at least 98%, d.s.b., commercialized byHayashibara Shoji, Inc., Okayama, one part by weight of α,β-trehalosewith a purity of at least 95%, d.s.b., one part by weight ofβ,β-trehalose, and three parts by weight of pullulan were tabletted witha tabletting machine with a punch of 6 mm in diameter to obtain tablets,about 300 mg each.

The product has an adequate strength because of its pullulan, and it canbe advantageously used as a health food that acts on epithelial cells ofmammalian small intestinal mucosae, controls the production of OPG,effectively inhibits the formation of osteoclast relating to osteopathy,and maintains/promotes the health of bone, when orally administered tomammals.

INDUSTRIAL APPLICABILITY

As explained above, the agent for controlling the production of OPG,which comprises a saccharide as an effective ingredient, has anadvantageous feature that effectively controls the formation ofosteoclast. Since the agent comprises a saccharide(s) as an effectiveingredient, it has a satisfactory flavor and taste, and it can be easilytaken or administered to living bodies without causing any inconveniencethrough oral and incubational administration routes, moderately acts onepithelial cells of mammalian small intestinal mucosae, and controls theproduction of OPG in the cells. As a result, the agent of the presentinvention can be advantageously used to effectively control theformation of osteoclast, which closely relates to bone diseases, andused to maintain/promote the health of bone.

The method for screening substances capable of controlling theproduction of OPG according to the present invention enables to screenpotential substances to be used in the agent of the present invention aseffective ingredients extensively, promptly, and easily.

The present invention with such outstanding functions and effects is asignificant invention that greatly contributes to this art.

1. An agent for controlling the production of osteoprotegerin, whichcomprises a saccharide as an effective ingredient.
 2. The agent of claim1, wherein said saccharide is one or more members selected from thegroup consisting of D-glucose, maltose, sucrose, α,α-trehalose,α,β-trehalose, and β,β-trehalose.
 3. The agent of claim 1, which furthercontains a calcium preparation.
 4. The agent of claim 1, which acts onepithelial cells of mammalian small intestinal mucosae and controls theproduction of osteoprotegerin in the cells.
 5. The agent of claim 1,which is in an orally or intubationally administrable form.
 6. A methodfor screening a substance capable of controlling the production ofosteoprotegerin, which comprises the steps of: culturing an establishedcell line capable of producing osteoprotegerin in a nutrient culturemedium; adding a test sample to the cell culture; culturing the cells inthe presence of the test sample to produce osteoprotegerin; assaying theproduction level of osteoprotegerin in the culture supernatant; andjudging the test sample to be a positive substance that can control theproduction of osteoprotegerin when the production level ofosteoprotegerin for the test sample is higher or lower than that for acontrol system.
 7. The method of claim 6, wherein said established cellline is MG-63 cells, ATCC CRL 1427; or FHs 74 Int cells, ATCC CCL 241.8. An agent for controlling the production of osteoprotegerin, whichcomprises as an effective ingredient a substance capable of controllingthe production of osteoprotegerin, said substance having been judgedpositive by the screening method of claim 6.